We propose to use new methods to study proteolysis of neurofilament proteins (NFPs) in an animal model system and in normal and Alzheimer's diseased (AD) human brains. Since neurofibrillary tangles, a hallmark of AD neurons, contain structures (paired helical filaments (PHFs) that possibly represent abnormal neurofilaments, these studies may lead to an understanding of how PHFs originate. We have developed new methods for analyzing proteolytic mechanisms in vitro and in vivo specifically within retinal ganglion cell (RGC) neurons that are well suited to studying neurofilament (and tubulin) proteolysis, transport and modification. Our initial observations indicate that the major NFPs (ca. 200K, 140K and 70K in mouse) may be comprised of multiple protein subcomponents within each size class and that more than one proteinase in RGC neurons is active toward normal NFPs. We propose of characterize NFPs and subcomponents in the RGC neuron in terms of their (1) transport, (2) fate within different neuronal compartments (perikaryon, axon, synapse), (3) modification before and during transport (phosphorylation, proteolytic sculpturing), (4) relative rates of degradation in vivo using double-isotope techniques, and (5) relative susceptibility in situ to individual proteinases (at least 4 presently identified) within the RGC neuron. One-dimensional SDS-polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis, peptide fingerprinting, and immunochemical techniques will be used. Using intact microslices of human postmortem cortex in which anatomic structure is maintained, we will identify proteinases that act on NFPs within neurons and quantitate the relative susceptibility of NFPs to each of these enzymes in situ. NFP proteolysis in AD cortical microslices displaying large numbers of PHFs will then be compared. Using a new micromethod, we will purify NFPs from discrete AD brain regions especially rich in PHFs and study their degradation by calcium-activated neutral proteinase, cathepsin B, cathepsin D, and neutral endopeptidase, each purified form normal human cortex. Alterations in the degradative rates or the nature of degradation products (compared to normal control NFPs) will be sought as an indication of defects in NFP structure in Alzheimer's disease.